The way organisms respond to radiation exposure is important since induced DNA lesions can lead to death, mutation or cancer. Previous efforts have focused on fission yeast S. pombe rad9, a gene that promotes gamma-ray resistance, UV-resistance, resistance to the DNA replication inhibitor hydroxyurea, and regulates the associated cell cycle checkpoints. We identified human (HRAD9) and mouse (Mrad9) orthologues, and the corresponding cDNAs were found to partially complement several defects demonstrated by rad9::ura4+ yeast. Furthermore, we found that HRAD9 protein binds the checkpoint proteins HHUS1 and HRAD1 at its C-terminal region, and contains a BH3-like domain at its N-terminal region that can bind the anti-apoptotic proteins BCL-2 and BCL-xL, and can cause apoptosis when overexpressed. We also found that this multifunctional protein can bind p53 and co-regulate p21. The major focus of this proposal builds on and extends a large amount of data accrued by us using yeast and mammalian systems. Specifically, we will make use of Mrad9 knockout cells constructed during the previous funding period to address well-defined hypotheses designed to elucidate Mrad9 function, and thus explain molecular mechanisms involved in the cellular response to DNA damage. These hypotheses include: 1) Mutations in the gene cause sensitivity to DNA damage at least in part because of defects in base excision repair, not just in cell cycle checkpoints; 2) Mrad9 regulates genomic stability in the presence or absence of exogenous DNA damaging agents, and it is mediated through the action of recombination proteins; 3) A newly discovered structurally and functionally related paralog of Rad9, called Mrad9B (HRAD9B), also plays important roles in mediating resistance to DNA damage and maintaining genomic stability, and the activities of the two related proteins are partially redundant. Experimental approacheswill address these hypotheses through the molecular, cellular and biochemical characterizationof Mrad9 mutant cells, and cells also altered in the Mrad9B paralog. These studies will examine Mrad9 function from molecular to cellular levels, define the structural basis for Mrad9 activity, and could impact on radiotherapy as well as on understanding genetic susceptibility to DNA damage.